quantitative real time pcr analysis total rna  (Promega)

 
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    Structured Review

    Promega quantitative real time pcr analysis total rna
    Overexpression of human AGGF1 in a mouse hindlimb ischemia model does not affect the expression level of VEGF . Seven days after delivery of AGGF1 expression plasmid DNA (empty vector as control), mice were sacrificed and gastrocnemius muscle tissues were excised and used for isolation of total <t>RNA</t> and follow-up real time <t>RT-PCR</t> analysis of the mouse VEGF gene. Note that VEGF expression did not show any difference between AGGF1 and control ( P > 0.05).
    Quantitative Real Time Pcr Analysis Total Rna, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Price from $9.99 to $1999.99
    quantitative real time pcr analysis total rna - by Bioz Stars, 2020-08
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    1) Product Images from "Angiogenic Factor AGGF1 Promotes Therapeutic Angiogenesis in a Mouse Limb Ischemia Model"

    Article Title: Angiogenic Factor AGGF1 Promotes Therapeutic Angiogenesis in a Mouse Limb Ischemia Model

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0046998

    Overexpression of human AGGF1 in a mouse hindlimb ischemia model does not affect the expression level of VEGF . Seven days after delivery of AGGF1 expression plasmid DNA (empty vector as control), mice were sacrificed and gastrocnemius muscle tissues were excised and used for isolation of total RNA and follow-up real time RT-PCR analysis of the mouse VEGF gene. Note that VEGF expression did not show any difference between AGGF1 and control ( P > 0.05).
    Figure Legend Snippet: Overexpression of human AGGF1 in a mouse hindlimb ischemia model does not affect the expression level of VEGF . Seven days after delivery of AGGF1 expression plasmid DNA (empty vector as control), mice were sacrificed and gastrocnemius muscle tissues were excised and used for isolation of total RNA and follow-up real time RT-PCR analysis of the mouse VEGF gene. Note that VEGF expression did not show any difference between AGGF1 and control ( P > 0.05).

    Techniques Used: Over Expression, Expressing, Plasmid Preparation, Mouse Assay, Isolation, Quantitative RT-PCR

    Successful overexpression of AGGF1 mRNA and protein in skeletal muscle by gene transfer using a plasmid-based delivery system. A) Real time PCR analysis for expression of human AGGF1 mRNA derived from an AGGF1 expression plasmid injected into the gastrocnemius muscle in a mouse ischemic hindlimb model. The RT-PCR primers are specific to human AGGF1 and not able to amplify the endogenous mouse AGGF1 mRNA. Time points are in days after the injection of plasmid DNA. Injection of the empty vector DNA served as negative control. The data are shown as the amount of plasmid-derived human AGGF1 mRNA normalized to control GAPDH levels. To avoid the contamination of plasmid DNA, RNA samples were treated with DNase. In addition, RT-PCR analysis with RNA samples without reverse transcription did not yield any product. B ) Expression of AGGF1 protein in gastrocnemius muscles by Western blot analysis at time points of 7, 14, 28 days after plasmid DNA was injected. The anti-AGGF1 antibody recognizes both human AGGF1 derived from the injected plasmid and endogenous mouse AGGF1 protein. C ) The images from Western blot analysis in B ) were scanned, quantified, and plotted. The intensity of AGGF1 signal was normalized to the signal of control β-actin. D ) Immunohistochemical analysis of the sections of ischemic hindlimb muscle from mice injected with an empty vector (Control) and with an AGGF1 expression plasmid (AGGF1) with an anti-AGGF1 antibody seven days later by injection of plasmid DNA. *, P
    Figure Legend Snippet: Successful overexpression of AGGF1 mRNA and protein in skeletal muscle by gene transfer using a plasmid-based delivery system. A) Real time PCR analysis for expression of human AGGF1 mRNA derived from an AGGF1 expression plasmid injected into the gastrocnemius muscle in a mouse ischemic hindlimb model. The RT-PCR primers are specific to human AGGF1 and not able to amplify the endogenous mouse AGGF1 mRNA. Time points are in days after the injection of plasmid DNA. Injection of the empty vector DNA served as negative control. The data are shown as the amount of plasmid-derived human AGGF1 mRNA normalized to control GAPDH levels. To avoid the contamination of plasmid DNA, RNA samples were treated with DNase. In addition, RT-PCR analysis with RNA samples without reverse transcription did not yield any product. B ) Expression of AGGF1 protein in gastrocnemius muscles by Western blot analysis at time points of 7, 14, 28 days after plasmid DNA was injected. The anti-AGGF1 antibody recognizes both human AGGF1 derived from the injected plasmid and endogenous mouse AGGF1 protein. C ) The images from Western blot analysis in B ) were scanned, quantified, and plotted. The intensity of AGGF1 signal was normalized to the signal of control β-actin. D ) Immunohistochemical analysis of the sections of ischemic hindlimb muscle from mice injected with an empty vector (Control) and with an AGGF1 expression plasmid (AGGF1) with an anti-AGGF1 antibody seven days later by injection of plasmid DNA. *, P

    Techniques Used: Over Expression, Plasmid Preparation, Real-time Polymerase Chain Reaction, Expressing, Derivative Assay, Injection, Reverse Transcription Polymerase Chain Reaction, Negative Control, Western Blot, Immunohistochemistry, Mouse Assay

    2) Product Images from "Promotion of flavonoid biosynthesis in leaves and calli of ornamental crabapple (Malus sp.) by high carbon to nitrogen ratios"

    Article Title: Promotion of flavonoid biosynthesis in leaves and calli of ornamental crabapple (Malus sp.) by high carbon to nitrogen ratios

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2015.00673

    Relative expression profiles of flavonoid/anthocyanin biosynthetic and regulatory genes in leaves (A) and calli (B) of crabapple Malus sp. cultivars grown under various C/N conditions . Real-time PCR was used to analyze the expression levels of MYB10 , CHS , F3H , F3’H , DFR , ANS , UFGT , and FLS . All real time-PCR reactions were normalized using the Ct value corresponding to a Malus crabapple 18S ribosomal RNA gene (DQ341382). Samples referred to on the x axis: (1) 30C/60N, (2) 90C/60N, (3) 150C/60N, (4) 90C/100N, and (5) 90C/20N. Error bars correspond to the SEM ± SE of three replicate analyses. Different letters above the bars indicate significantly different values ( P
    Figure Legend Snippet: Relative expression profiles of flavonoid/anthocyanin biosynthetic and regulatory genes in leaves (A) and calli (B) of crabapple Malus sp. cultivars grown under various C/N conditions . Real-time PCR was used to analyze the expression levels of MYB10 , CHS , F3H , F3’H , DFR , ANS , UFGT , and FLS . All real time-PCR reactions were normalized using the Ct value corresponding to a Malus crabapple 18S ribosomal RNA gene (DQ341382). Samples referred to on the x axis: (1) 30C/60N, (2) 90C/60N, (3) 150C/60N, (4) 90C/100N, and (5) 90C/20N. Error bars correspond to the SEM ± SE of three replicate analyses. Different letters above the bars indicate significantly different values ( P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

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    Amplification:

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    Article Snippet: .. Reverse transcription-PCR (RT-PCR) and quantitative real-time RT-PCR Total RNA was reverse-transcribed and amplified using reverse transcription and PCR kits, respectively (Promega Corp., Madison, WI, U.S.). .. Real-time RT-PCR was performed by Bio-Rad IQ5 (Bio-Rad, U.S.).

    Quantitative RT-PCR:

    Article Title: Effects of THAP11 on Erythroid Differentiation and Megakaryocytic Differentiation of K562 Cells
    Article Snippet: .. Reverse transcription-PCR (RT-PCR) and quantitative real-time RT-PCR Total RNA was reverse-transcribed and amplified using reverse transcription and PCR kits, respectively (Promega Corp., Madison, WI, U.S.). .. Real-time RT-PCR was performed by Bio-Rad IQ5 (Bio-Rad, U.S.).

    Real-time Polymerase Chain Reaction:

    Article Title: Avian Tembusu virus infection effectively triggers host innate immune response through MDA5 and TLR3-dependent signaling pathways
    Article Snippet: .. Equal amounts of RNA (4 μg) was used for reverse-transcription PCR to prepare cDNA using M-MLV Reverse Transcriptase (Promega, USA), followed by PCR using rTaq DNA polymerase and quantitative real-time PCR using TransStart Green qPCR SuperMix (TransGen). .. The primers for ATMUV and chicken IFN-β, IFN-λ gene were designed using the Primer 5 software; other sequences of the primers used in this study have been described previously [ – ].

    Article Title: Cilia-related protein SPEF2 regulates osteoblast differentiation
    Article Snippet: .. Real-time PCR (RT-PCR) For analysis of gene expression with RT-PCR the total RNA was reverse transcribed with random primers and an RT-PCR kit (ImProm-II Reverse Transcription System, Promega) according to the manufacturer’s instructions. .. Produced cDNA was amplified by using gene specific primers listed in Supplemental Table .

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Aspergillus fumigatus Survival in Alkaline and Extreme Zinc-Limiting Environments Relies on the Induction of a Zinc Homeostasis System Encoded by the zrfC and aspf2 Genes ▿ Genes ▿ †
    Article Snippet: .. The cDNA of zrfC was obtained by reverse transcription (RT)-PCR using oligonucleotides JA299 (for retrotranscription) and JA60 and JA54 (both for PCR), and a 1.6-kb cDNA fragment was subcloned into pGEM-T Easy (Promega) to generate plasmid pZRF30 and sequenced. .. The cDNA of aspf2 was obtained by RT-PCR using oligonucleotides JA8 (for retrotranscription) and JA166 and JA167 (both for PCR), and a 0.96-kb cDNA fragment was subcloned into pGEM-T Easy to generate plasmid pASPF23 and sequenced.

    Article Title: Restricted chromosomal silencing in nucleolar dominance
    Article Snippet: .. Reverse transcription (RT)-PCR was performed by using RNA that had been treated with RQ1 DNase I (Promega) to eliminate any contaminating genomic DNA. .. Total RNA was used in all cases except in the case of F16N15.13 , for which poly(A)+ RNA was isolated on oligo(dT) paramagnetic beads according to the manufacturer's instructions (Dynal, Great Neck, NY).

    Article Title: Effects of THAP11 on Erythroid Differentiation and Megakaryocytic Differentiation of K562 Cells
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    Article Title: Analysis of Transcription of the Staphylococcus aureus Aerobic Class Ib and Anaerobic Class III Ribonucleotide Reductase Genes in Response to Oxygen
    Article Snippet: .. For reverse transcription (RT)-PCR and primer extension, residual DNA was removed by treatment with RQ1 RNase-free DNase (Promega). .. RNA concentrations were determined by A 260 measurements, and RNA integrity was analyzed by agarose/formaldehyde gel electrophoresis ( ).

    Article Title: Virucidal nano-perforator of viral membrane trapping viral RNAs in the endosome
    Article Snippet: .. To confirm the release of vRNPs, reverse transcription (RT)-PCR was carried out using M-MLV reverse transcriptase (M1705, Promega, Madison, WI, USA) according to the manufacturer’s protocol using primers for the viral M gene of A/Puerto Rico/8/34; 5′-TGCACTTTGACATTGTGGATTCTTG-3′ (M_FW) and 5′-CCCTCATAGACTTTGGCACTCC-3′ (M_BW) . .. The coding region of the M sequence in the RT product was amplified with the described primers by using rTaq Plus 5 × PCR Master Mix (ELPIS-BIOTECH, Inc., Daejeon, Korea) according to the manufacturer’s protocol under the following thermal cycling conditions: 30 cycles of 95 °C for 10 s, 56 °C for 10 s, and 72 °C for 10 s. PCR products were separated by electrophoresis at 135 V for 20 min on a 1% agarose (Cosmogenetech, Inc., Korea) gel and visualised with a UV transilluminator.

    Article Title: Cilia-related protein SPEF2 regulates osteoblast differentiation
    Article Snippet: .. Real-time PCR (RT-PCR) For analysis of gene expression with RT-PCR the total RNA was reverse transcribed with random primers and an RT-PCR kit (ImProm-II Reverse Transcription System, Promega) according to the manufacturer’s instructions. .. Produced cDNA was amplified by using gene specific primers listed in Supplemental Table .

    Expressing:

    Article Title: Cilia-related protein SPEF2 regulates osteoblast differentiation
    Article Snippet: .. Real-time PCR (RT-PCR) For analysis of gene expression with RT-PCR the total RNA was reverse transcribed with random primers and an RT-PCR kit (ImProm-II Reverse Transcription System, Promega) according to the manufacturer’s instructions. .. Produced cDNA was amplified by using gene specific primers listed in Supplemental Table .

    Polymerase Chain Reaction:

    Article Title: Aspergillus fumigatus Survival in Alkaline and Extreme Zinc-Limiting Environments Relies on the Induction of a Zinc Homeostasis System Encoded by the zrfC and aspf2 Genes ▿ Genes ▿ †
    Article Snippet: .. The cDNA of zrfC was obtained by reverse transcription (RT)-PCR using oligonucleotides JA299 (for retrotranscription) and JA60 and JA54 (both for PCR), and a 1.6-kb cDNA fragment was subcloned into pGEM-T Easy (Promega) to generate plasmid pZRF30 and sequenced. .. The cDNA of aspf2 was obtained by RT-PCR using oligonucleotides JA8 (for retrotranscription) and JA166 and JA167 (both for PCR), and a 0.96-kb cDNA fragment was subcloned into pGEM-T Easy to generate plasmid pASPF23 and sequenced.

    Article Title: Avian Tembusu virus infection effectively triggers host innate immune response through MDA5 and TLR3-dependent signaling pathways
    Article Snippet: .. Equal amounts of RNA (4 μg) was used for reverse-transcription PCR to prepare cDNA using M-MLV Reverse Transcriptase (Promega, USA), followed by PCR using rTaq DNA polymerase and quantitative real-time PCR using TransStart Green qPCR SuperMix (TransGen). .. The primers for ATMUV and chicken IFN-β, IFN-λ gene were designed using the Primer 5 software; other sequences of the primers used in this study have been described previously [ – ].

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    Article Snippet: .. Reverse transcription-PCR (RT-PCR) and quantitative real-time RT-PCR Total RNA was reverse-transcribed and amplified using reverse transcription and PCR kits, respectively (Promega Corp., Madison, WI, U.S.). .. Real-time RT-PCR was performed by Bio-Rad IQ5 (Bio-Rad, U.S.).

    Plasmid Preparation:

    Article Title: Aspergillus fumigatus Survival in Alkaline and Extreme Zinc-Limiting Environments Relies on the Induction of a Zinc Homeostasis System Encoded by the zrfC and aspf2 Genes ▿ Genes ▿ †
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    Promega quantitative real time pcr rt qpcr analysis total rna
    Vibrio campbellii -exposed parental generation and their unexposed progenies showed increased expression of hsp70 and hmgb1 genes. See Fig. 9 for explanation of the experimental groups. Total <t>RNA</t> extracted from F0 to F3 juveniles, reared under common garden conditions, was analyzed for expression of ( A ) hsp70 and ( B ) hmgb1 genes by <t>qPCR</t> assay. At each generation, the expression of hsp70 or hmgb1 gene in the control group was set at 1.0 and all other data points were normalized accordingly using the equation of Pfaffl et al. (2002) 52 . The actin gene was used as an internal control. Error bars represent the standard errors of three biological replicates. Significant differences between the treatment and control at respective generation are indicated by *(P
    Quantitative Real Time Pcr Rt Qpcr Analysis Total Rna, supplied by Promega, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/quantitative real time pcr rt qpcr analysis total rna/product/Promega
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    92
    Promega quantitative real time pcr analysis total rna
    Overexpression of human AGGF1 in a mouse hindlimb ischemia model does not affect the expression level of VEGF . Seven days after delivery of AGGF1 expression plasmid DNA (empty vector as control), mice were sacrificed and gastrocnemius muscle tissues were excised and used for isolation of total <t>RNA</t> and follow-up real time <t>RT-PCR</t> analysis of the mouse VEGF gene. Note that VEGF expression did not show any difference between AGGF1 and control ( P > 0.05).
    Quantitative Real Time Pcr Analysis Total Rna, supplied by Promega, used in various techniques. Bioz Stars score: 92/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/quantitative real time pcr analysis total rna/product/Promega
    Average 92 stars, based on 13 article reviews
    Price from $9.99 to $1999.99
    quantitative real time pcr analysis total rna - by Bioz Stars, 2020-08
    92/100 stars
      Buy from Supplier

    92
    Promega quantitative real time pcr qrt pcr analysis total rna
    Developmental stage- and sex-specific expression of immune-related genes in N. lugens Total <t>RNA</t> was extracted from eggs, 2nd instar nymphs, 5th instar nymphs, female adults and male adults, individually. First-strand cDNA (20 ng) was analyzed in each <t>qRT-PCR</t> reaction. The reactions were performed with specific primers for amplifying ( A ) PGRP/GRP genes; ( B ) Toll genes; ( C ) CLIP genes; and ( D ) immune effector genes. The relative expression levels of each gene in each developmental stage or sex were normalized using the N. lugens 18 s rRNA threshold cycle (Ct) values that were obtained from reactions run on the same plate. In each assay, the expression level was normalized to the lowest expression level, which was arbitrarily set at one. Three technical replication (n=3) was conducted and the ΔΔCt method was used to measure the relative transcript levels in each treated sample.
    Quantitative Real Time Pcr Qrt Pcr Analysis Total Rna, supplied by Promega, used in various techniques. Bioz Stars score: 92/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/quantitative real time pcr qrt pcr analysis total rna/product/Promega
    Average 92 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    quantitative real time pcr qrt pcr analysis total rna - by Bioz Stars, 2020-08
    92/100 stars
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    Vibrio campbellii -exposed parental generation and their unexposed progenies showed increased expression of hsp70 and hmgb1 genes. See Fig. 9 for explanation of the experimental groups. Total RNA extracted from F0 to F3 juveniles, reared under common garden conditions, was analyzed for expression of ( A ) hsp70 and ( B ) hmgb1 genes by qPCR assay. At each generation, the expression of hsp70 or hmgb1 gene in the control group was set at 1.0 and all other data points were normalized accordingly using the equation of Pfaffl et al. (2002) 52 . The actin gene was used as an internal control. Error bars represent the standard errors of three biological replicates. Significant differences between the treatment and control at respective generation are indicated by *(P

    Journal: Scientific Reports

    Article Title: Probing the phenomenon of trained immunity in invertebrates during a transgenerational study, using brine shrimp Artemia as a model system

    doi: 10.1038/srep21166

    Figure Lengend Snippet: Vibrio campbellii -exposed parental generation and their unexposed progenies showed increased expression of hsp70 and hmgb1 genes. See Fig. 9 for explanation of the experimental groups. Total RNA extracted from F0 to F3 juveniles, reared under common garden conditions, was analyzed for expression of ( A ) hsp70 and ( B ) hmgb1 genes by qPCR assay. At each generation, the expression of hsp70 or hmgb1 gene in the control group was set at 1.0 and all other data points were normalized accordingly using the equation of Pfaffl et al. (2002) 52 . The actin gene was used as an internal control. Error bars represent the standard errors of three biological replicates. Significant differences between the treatment and control at respective generation are indicated by *(P

    Article Snippet: RNA extraction and quantitative real-time PCR (RT-qPCR) analysis Total RNA was extracted from the Artemia samples using the SV total RNA isolation kit (Promega, Belgium).

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    Overexpression of human AGGF1 in a mouse hindlimb ischemia model does not affect the expression level of VEGF . Seven days after delivery of AGGF1 expression plasmid DNA (empty vector as control), mice were sacrificed and gastrocnemius muscle tissues were excised and used for isolation of total RNA and follow-up real time RT-PCR analysis of the mouse VEGF gene. Note that VEGF expression did not show any difference between AGGF1 and control ( P > 0.05).

    Journal: PLoS ONE

    Article Title: Angiogenic Factor AGGF1 Promotes Therapeutic Angiogenesis in a Mouse Limb Ischemia Model

    doi: 10.1371/journal.pone.0046998

    Figure Lengend Snippet: Overexpression of human AGGF1 in a mouse hindlimb ischemia model does not affect the expression level of VEGF . Seven days after delivery of AGGF1 expression plasmid DNA (empty vector as control), mice were sacrificed and gastrocnemius muscle tissues were excised and used for isolation of total RNA and follow-up real time RT-PCR analysis of the mouse VEGF gene. Note that VEGF expression did not show any difference between AGGF1 and control ( P > 0.05).

    Article Snippet: Quantitative Real-time PCR Analysis Total RNA samples were extracted from gastrocnemius muscles using TRIzol (Invitrogene), precipitated with isopropanol, and treated with RQ1 RNase-Free DNase (Promega) to eliminate DNA contamination.

    Techniques: Over Expression, Expressing, Plasmid Preparation, Mouse Assay, Isolation, Quantitative RT-PCR

    Successful overexpression of AGGF1 mRNA and protein in skeletal muscle by gene transfer using a plasmid-based delivery system. A) Real time PCR analysis for expression of human AGGF1 mRNA derived from an AGGF1 expression plasmid injected into the gastrocnemius muscle in a mouse ischemic hindlimb model. The RT-PCR primers are specific to human AGGF1 and not able to amplify the endogenous mouse AGGF1 mRNA. Time points are in days after the injection of plasmid DNA. Injection of the empty vector DNA served as negative control. The data are shown as the amount of plasmid-derived human AGGF1 mRNA normalized to control GAPDH levels. To avoid the contamination of plasmid DNA, RNA samples were treated with DNase. In addition, RT-PCR analysis with RNA samples without reverse transcription did not yield any product. B ) Expression of AGGF1 protein in gastrocnemius muscles by Western blot analysis at time points of 7, 14, 28 days after plasmid DNA was injected. The anti-AGGF1 antibody recognizes both human AGGF1 derived from the injected plasmid and endogenous mouse AGGF1 protein. C ) The images from Western blot analysis in B ) were scanned, quantified, and plotted. The intensity of AGGF1 signal was normalized to the signal of control β-actin. D ) Immunohistochemical analysis of the sections of ischemic hindlimb muscle from mice injected with an empty vector (Control) and with an AGGF1 expression plasmid (AGGF1) with an anti-AGGF1 antibody seven days later by injection of plasmid DNA. *, P

    Journal: PLoS ONE

    Article Title: Angiogenic Factor AGGF1 Promotes Therapeutic Angiogenesis in a Mouse Limb Ischemia Model

    doi: 10.1371/journal.pone.0046998

    Figure Lengend Snippet: Successful overexpression of AGGF1 mRNA and protein in skeletal muscle by gene transfer using a plasmid-based delivery system. A) Real time PCR analysis for expression of human AGGF1 mRNA derived from an AGGF1 expression plasmid injected into the gastrocnemius muscle in a mouse ischemic hindlimb model. The RT-PCR primers are specific to human AGGF1 and not able to amplify the endogenous mouse AGGF1 mRNA. Time points are in days after the injection of plasmid DNA. Injection of the empty vector DNA served as negative control. The data are shown as the amount of plasmid-derived human AGGF1 mRNA normalized to control GAPDH levels. To avoid the contamination of plasmid DNA, RNA samples were treated with DNase. In addition, RT-PCR analysis with RNA samples without reverse transcription did not yield any product. B ) Expression of AGGF1 protein in gastrocnemius muscles by Western blot analysis at time points of 7, 14, 28 days after plasmid DNA was injected. The anti-AGGF1 antibody recognizes both human AGGF1 derived from the injected plasmid and endogenous mouse AGGF1 protein. C ) The images from Western blot analysis in B ) were scanned, quantified, and plotted. The intensity of AGGF1 signal was normalized to the signal of control β-actin. D ) Immunohistochemical analysis of the sections of ischemic hindlimb muscle from mice injected with an empty vector (Control) and with an AGGF1 expression plasmid (AGGF1) with an anti-AGGF1 antibody seven days later by injection of plasmid DNA. *, P

    Article Snippet: Quantitative Real-time PCR Analysis Total RNA samples were extracted from gastrocnemius muscles using TRIzol (Invitrogene), precipitated with isopropanol, and treated with RQ1 RNase-Free DNase (Promega) to eliminate DNA contamination.

    Techniques: Over Expression, Plasmid Preparation, Real-time Polymerase Chain Reaction, Expressing, Derivative Assay, Injection, Reverse Transcription Polymerase Chain Reaction, Negative Control, Western Blot, Immunohistochemistry, Mouse Assay

    NFAT5 regulates REDD1 expression in HT29 cells. (A and B) HT29 cells were transfected with NFAT5 or NTC siRNA. After 48-h incubation, transfected cells were lysed, total RNA was extracted, and real-time RT PCR was performed for analysis of REDD1 (A) and NFAT5 and NFATc1, NFATc2, NFATc3, and NFATc4 mRNA expression (B). (Data represent mean ± SD; *, p

    Journal: Molecular Biology of the Cell

    Article Title: Nuclear factor of activated T-cells 5 increases intestinal goblet cell differentiation through an mTOR/Notch signaling pathway

    doi: 10.1091/mbc.E14-05-0998

    Figure Lengend Snippet: NFAT5 regulates REDD1 expression in HT29 cells. (A and B) HT29 cells were transfected with NFAT5 or NTC siRNA. After 48-h incubation, transfected cells were lysed, total RNA was extracted, and real-time RT PCR was performed for analysis of REDD1 (A) and NFAT5 and NFATc1, NFATc2, NFATc3, and NFATc4 mRNA expression (B). (Data represent mean ± SD; *, p

    Article Snippet: Quantitative real time RT-PCR analysis Total RNA was extracted and DNase treated (RQ1; Promega, Madison, WI).

    Techniques: Expressing, Transfection, Incubation, Quantitative RT-PCR

    Regulation of MUC2 mRNA expression by mTORC1/Notch signaling pathway. (A) HT29 cells were transfected with NTC siRNA or siRNA targeting REDD1. (B) HT29 cells were treated with 100 nM rapamycin for 24 h. Total protein was extracted, and Western blotting was performed using anti-NICD, REDD1, anti–p-Ser-6, anti-Ser-6, Hes1, and anti–β-actin antibodies. NICD and Hes1 signals from three separate experiments were quantitated densitometrically and expressed as fold change with respect to β-actin. (C) HT29 cells were treated with 100 nM rapamycin for 24 h; total RNA was extracted and MUC2 mRNA levels were determined by real-time RT-PCR. (Data represent mean ± SD; *, p

    Journal: Molecular Biology of the Cell

    Article Title: Nuclear factor of activated T-cells 5 increases intestinal goblet cell differentiation through an mTOR/Notch signaling pathway

    doi: 10.1091/mbc.E14-05-0998

    Figure Lengend Snippet: Regulation of MUC2 mRNA expression by mTORC1/Notch signaling pathway. (A) HT29 cells were transfected with NTC siRNA or siRNA targeting REDD1. (B) HT29 cells were treated with 100 nM rapamycin for 24 h. Total protein was extracted, and Western blotting was performed using anti-NICD, REDD1, anti–p-Ser-6, anti-Ser-6, Hes1, and anti–β-actin antibodies. NICD and Hes1 signals from three separate experiments were quantitated densitometrically and expressed as fold change with respect to β-actin. (C) HT29 cells were treated with 100 nM rapamycin for 24 h; total RNA was extracted and MUC2 mRNA levels were determined by real-time RT-PCR. (Data represent mean ± SD; *, p

    Article Snippet: Quantitative real time RT-PCR analysis Total RNA was extracted and DNase treated (RQ1; Promega, Madison, WI).

    Techniques: Expressing, Transfection, Western Blot, Quantitative RT-PCR

    NFAT5 regulation of MUC2 mRNA expression. (A) HT29 and Caco-2 cells were transfected with NTC siRNA or siRNA targeting NFAT5. (B) HCT116 and Caco-2 cells were transfected with control vector or NFAT5 plasmid. After 48-h incubation, total RNA was extracted, and MUC2 mRNA levels were determined by real-time RT-PCR. (Data represent mean ± SD; *, p

    Journal: Molecular Biology of the Cell

    Article Title: Nuclear factor of activated T-cells 5 increases intestinal goblet cell differentiation through an mTOR/Notch signaling pathway

    doi: 10.1091/mbc.E14-05-0998

    Figure Lengend Snippet: NFAT5 regulation of MUC2 mRNA expression. (A) HT29 and Caco-2 cells were transfected with NTC siRNA or siRNA targeting NFAT5. (B) HCT116 and Caco-2 cells were transfected with control vector or NFAT5 plasmid. After 48-h incubation, total RNA was extracted, and MUC2 mRNA levels were determined by real-time RT-PCR. (Data represent mean ± SD; *, p

    Article Snippet: Quantitative real time RT-PCR analysis Total RNA was extracted and DNase treated (RQ1; Promega, Madison, WI).

    Techniques: Expressing, Transfection, Plasmid Preparation, Incubation, Quantitative RT-PCR

    Developmental stage- and sex-specific expression of immune-related genes in N. lugens Total RNA was extracted from eggs, 2nd instar nymphs, 5th instar nymphs, female adults and male adults, individually. First-strand cDNA (20 ng) was analyzed in each qRT-PCR reaction. The reactions were performed with specific primers for amplifying ( A ) PGRP/GRP genes; ( B ) Toll genes; ( C ) CLIP genes; and ( D ) immune effector genes. The relative expression levels of each gene in each developmental stage or sex were normalized using the N. lugens 18 s rRNA threshold cycle (Ct) values that were obtained from reactions run on the same plate. In each assay, the expression level was normalized to the lowest expression level, which was arbitrarily set at one. Three technical replication (n=3) was conducted and the ΔΔCt method was used to measure the relative transcript levels in each treated sample.

    Journal: BMC Genomics

    Article Title: The genome- and transcriptome-wide analysis of innate immunity in the brown planthopper, Nilaparvata lugens

    doi: 10.1186/1471-2164-14-160

    Figure Lengend Snippet: Developmental stage- and sex-specific expression of immune-related genes in N. lugens Total RNA was extracted from eggs, 2nd instar nymphs, 5th instar nymphs, female adults and male adults, individually. First-strand cDNA (20 ng) was analyzed in each qRT-PCR reaction. The reactions were performed with specific primers for amplifying ( A ) PGRP/GRP genes; ( B ) Toll genes; ( C ) CLIP genes; and ( D ) immune effector genes. The relative expression levels of each gene in each developmental stage or sex were normalized using the N. lugens 18 s rRNA threshold cycle (Ct) values that were obtained from reactions run on the same plate. In each assay, the expression level was normalized to the lowest expression level, which was arbitrarily set at one. Three technical replication (n=3) was conducted and the ΔΔCt method was used to measure the relative transcript levels in each treated sample.

    Article Snippet: Quantitative real-time PCR (qRT-PCR) analysis Total RNA was isolated from N. lugens specimens using the SV Total RNA Isolation System (Promega).

    Techniques: Expressing, Quantitative RT-PCR, Cross-linking Immunoprecipitation

    Responsive expressions to bacterial infection of immune-related genes in N. lugens nymphs. Fifth instar nymphs were microinjected with E. coli K12 or B. subtilis . Total RNA was extracted from the nymphs at the indicated times after injection. PBS-injected samples were used as controls. First-strand cDNA (20 ng) was analyzed in each real-time quantitative PCR reaction. The reactions were performed with specific primers for amplifying PGRP/GRP genes, immune effector genes and Toll genes. The relative expression levels of each gene at different time points were normalized using the N. lugens 18 s rRNA threshold cycle (Ct) values, which were obtained for reactions run on the same plate. In each assay, the expression level was normalized to the lowest expression level, which was arbitrarily set to one. Three technical replications (n=3) were conducted and the relative transcript levels at each time point were calculated using the ΔΔCt method. The E. coli K12- and B. subtilis injected samples are shown on the left (black) and right (dark gray), respectively. C refers to the PBS-injected control. 6, 12, and 24 h refer to RNA extracted from bacteria-injected nymphs at 6, 12, and 24 h p.i.

    Journal: BMC Genomics

    Article Title: The genome- and transcriptome-wide analysis of innate immunity in the brown planthopper, Nilaparvata lugens

    doi: 10.1186/1471-2164-14-160

    Figure Lengend Snippet: Responsive expressions to bacterial infection of immune-related genes in N. lugens nymphs. Fifth instar nymphs were microinjected with E. coli K12 or B. subtilis . Total RNA was extracted from the nymphs at the indicated times after injection. PBS-injected samples were used as controls. First-strand cDNA (20 ng) was analyzed in each real-time quantitative PCR reaction. The reactions were performed with specific primers for amplifying PGRP/GRP genes, immune effector genes and Toll genes. The relative expression levels of each gene at different time points were normalized using the N. lugens 18 s rRNA threshold cycle (Ct) values, which were obtained for reactions run on the same plate. In each assay, the expression level was normalized to the lowest expression level, which was arbitrarily set to one. Three technical replications (n=3) were conducted and the relative transcript levels at each time point were calculated using the ΔΔCt method. The E. coli K12- and B. subtilis injected samples are shown on the left (black) and right (dark gray), respectively. C refers to the PBS-injected control. 6, 12, and 24 h refer to RNA extracted from bacteria-injected nymphs at 6, 12, and 24 h p.i.

    Article Snippet: Quantitative real-time PCR (qRT-PCR) analysis Total RNA was isolated from N. lugens specimens using the SV Total RNA Isolation System (Promega).

    Techniques: Infection, Injection, Real-time Polymerase Chain Reaction, Expressing

    Tissue specificity of immune-related gene expression in N. lugens. Total RNA was individually extracted from the salivary gland, fat body, gut and the remaining carcass of 5th instar nymphs. First-strand cDNA (20 ng) was analyzed in each qRT-PCR reaction. The reactions were performed with specific primers used to amplify ( A ) PGRP/GRP genes; ( B ) Toll genes; ( C ) CLIP genes; and ( D ) immune effector genes. The relative expression levels of each gene in each tissue were normalized using the N. lugens 18 s rRNA threshold cycle (Ct) values which were obtained from reactions run on the same plate. In each assay, the expression level was normalized to the lowest expression level, which was arbitrarily set at one. Three technical replications (n=3) were conducted and the ΔΔCt method was used to measure the relative transcript levels in tissues.

    Journal: BMC Genomics

    Article Title: The genome- and transcriptome-wide analysis of innate immunity in the brown planthopper, Nilaparvata lugens

    doi: 10.1186/1471-2164-14-160

    Figure Lengend Snippet: Tissue specificity of immune-related gene expression in N. lugens. Total RNA was individually extracted from the salivary gland, fat body, gut and the remaining carcass of 5th instar nymphs. First-strand cDNA (20 ng) was analyzed in each qRT-PCR reaction. The reactions were performed with specific primers used to amplify ( A ) PGRP/GRP genes; ( B ) Toll genes; ( C ) CLIP genes; and ( D ) immune effector genes. The relative expression levels of each gene in each tissue were normalized using the N. lugens 18 s rRNA threshold cycle (Ct) values which were obtained from reactions run on the same plate. In each assay, the expression level was normalized to the lowest expression level, which was arbitrarily set at one. Three technical replications (n=3) were conducted and the ΔΔCt method was used to measure the relative transcript levels in tissues.

    Article Snippet: Quantitative real-time PCR (qRT-PCR) analysis Total RNA was isolated from N. lugens specimens using the SV Total RNA Isolation System (Promega).

    Techniques: Expressing, Quantitative RT-PCR, Cross-linking Immunoprecipitation